Age-related resistance to Pseudomonas syringae pv. tomato is associated with the transition to flowering in Arabidopsis and is effective against Peronospora parasitica

Abstract

As plants mature it has been observed that some become more resistant to normally virulent pathogens. The ability to manifest the Age-Related Resistance (ARR) response in Arabidopsis to Pseudomonas syringae pathovars tomato (Pst) coincided with the transition to flowering in plants both delayed and accelerated in the transition to flowering. ARR was also associated with a change in PR-1 gene expression, such that young plants expressed PR-1 abundantly at 3 days post inoculation (dpi) while mature plants expressed much less. The Arabidopsis ARR response requires SA accumulation via isochorismate synthase (ICS1) [24]. ICS1 was expressed one dpi with virulent and avirulent Pst in both young and mature plants. The ARR response was also effective versus avirulent Pst providing an additional 4-fold limitation in bacterial growth. Arabidopsis ARR was found to be ineffective against two necrotrophs, Erwinia carotovora subspecies carotovora (bacterium) and Botrytis cinerea (fungus) and one obligate biotroph, Erysiphe cichoracearum (fungus). However, mature wild type, SA-deficient sid2 and NahG plants supported little growth of the obligate biotrophic oomycete, Peronospora parasitica. Therefore ARR to P. parasitica appears to be SA-independent, however the level of ARR resistance was somewhat reduced in these mutants in some experiments. Thus, there may be numerous defence pathways that contribute to adult plant resistance in Arabidopsis.

Introduction

It has been observed that as some plant species mature, they become more resistant to normally virulent pathogens. This Age-related resistance (ARR) has been correlated with the transition to flowering in some cases or with senescence in others and also with the accumulation of secondary metabolites or defense proteins (reviewed in [28]). Whether ARR affords broad-spectrum resistance to unrelated pathogens has not been addressed in previous ARR studies [28]. However in a number of separate studies, mature tobacco plants were observed to become more resistant to two oomycetes, (Peronospora tabacina [43], Phytophthora parasitica [20]) and to tobacco mosaic virus (TMV [15], [44], [45]). Interestingly, resistance to P. tabacina correlated with the onset of senescence, while resistance to both P. parasitica and TMV correlated with the transition to flowering, suggesting that more than one ARR pathway may exist in tobacco. More recent studies have uncovered both a salicylic acid (SA)-dependent and independent ARR response in tobacco to P. parasitica at the transition to flowering stage [20], [21]. These results suggest that there may be numerous ARR pathways that contribute to resistance in adult plants.

Recently, ARR in Arabidopsis to Pseudomonas syringae pathovars. tomato (Pst) and maculicola (Psm) [24] was shown to be an unique defense response that occurs in a number of defense response mutants, including, npr1/nim1 [7], [11], etr1-4 [19], pad3-1 [17], [18] and eds7-1 [31]. These data strongly suggest that ARR is not a form of Systemic Acquired Resistance (SAR) or Induced Systemic Resistance and that the phytoalexin, camalexin is not required [24]. Additionally, there was no correlation between the ARR response and expression of the Pathogenesis Related-1 gene (PR-1), a molecular marker for the SAR response [35], [38] defined as a localized attack by a necrotizing pathogen which induces resistance in distant tissues to subsequent attack by a broad range of normally virulent pathogens (reviewed in [25]).

SA accumulation is required during many R gene-mediated resistance responses, as well as in basal defense to some pathogens and in the SAR response [10], [11], [36], [37] in both an NPR1-dependent and independent manner (reviewed in [32]). There is mounting evidence that SA acts as a signaling molecule during defense by inducing the translocation of NPR1, a key regulatory protein into the nucleus, where NPR1 interacts with TGA transcription factors which subsequently up-regulate defense genes like PR-1 [12], [14], [22], [23], [33], [46], [47]. Recent evidence also demonstrates that SA accumulation results in the reduction of the TGA1 factor allowing its interaction with NPR1 and subsequent promotion of TGA1 binding to the PR-1 promoter [13]. Rapid and abundant PR-1 gene expression, within one-day post inoculation (dpi), has been correlated with R gene-mediated resistance and SAR, while delayed expression (>1 dpi) of PR-1 has been observed during basal defense to virulent pathogens, including Pst [5], [48].

It is interesting to note that the relationship between PR-1 gene expression and SA accumulation in ARR is different than in R gene-mediated resistance, basal resistance or SAR. As discussed above, accumulation of SA activates NPR1 and the TGA factors leading to expression of PR-1. However, PR-1 gene expression is reduced in plants expressing ARR, even though SA accumulation is required. Kus et al. [24] demonstrated that the transgenic line NahG [10] and mutants, eds5/sid1 and sid2 [26], [27] are defective for the ARR response. These lines accumulate little SA and SID2 encodes isochorismate synthase (ICS1, [41]) suggesting that the ability to accumulate SA via the chorismate biosynthetic pathway is necessary for the ARR response. Recent evidence from our lab suggests that SA accumulates and acts in the intercellular space during ARR. Additionally, SA has in vitro anti-microbial activity against Pst suggesting that SA may play a different role during ARR, that of an anti-microbial agent [6].

In the Kus et al. study [24], the ARR response was not associated with senescence as the early senescence-associated marker, SAG-13 [39] was not expressed in mature plants (∼6 weeks of age) grown under short day conditions. Additionally, few inflorescence shoots were observed in mature plants exhibiting ARR, although a careful determination of the number of plants that had made the transition to flowering was not undertaken. Arabidopsis (ecotype Col-0) has been observed to begin flowering at 5 weeks of age under short day length conditions [29], hinting that ARR in Arabidopsis may be associated with the transition to flowering stage.

In this study, a number of questions were addressed. Does ARR in Arabidopsis provide protection to pathogens other than Pst and Psm? syringae pv. tomato (Pst) and maculicola? Moreover, if ARR is functional against other pathogens, is SA accumulation also required? To address this question, the Arabidopsis ARR response was tested against two necrotrophic pathogens, Erwinia carotovora subspecies carotovora (bacterium) and Botrytis cinerea (fungus). Both attack many plant species, including Arabidopsis, killing plant cells and releasing essential nutrients for pathogen growth [1], [30], [34]. As well, two obligate biotrophic pathogens, Erysiphe cichoracearum (fungus, [1] and Peronospora parasitica (oomycete, [9]), which obtain nutrients from living plant cells, were also tested in ARR assays. The question of whether ARR is associated with the transition to flowering was addressed by comparing the ARR response in plants grown under short day (SD) (delayed transition to flowering) versus long day (LD) (accelerated transition to flowering) conditions [29]. Early- and delayed-flowering mutants were not used to address this question as these mutants display pleiotropic phenotypes indicating that the corresponding genes affect many processes [3], [8], therefore, it would be difficult determine if ARR is associated with the transition to flowering or with some other pathway affected in these mutants.

In our previous work, PR-1 gene expression was reduced in mature (6 weeks post germination, wpg) compared to young (3–4 wpg) plants [24], suggesting that reduced PR-1 expression may be a molecular marker for ARR. Therefore, a detailed look at PR-1 expression during the plant life cycle at 3–6 wpg was performed to determine if reduced PR-1 gene expression correlated with plant age and the ability to manifest ARR. We also determined if the ARR response is effective versus avirulent Pst. Additionally, ICS1 gene expression, the penultimate gene of chorismate-derived SA biosynthesis [41], was also examined in SD plants exhibiting ARR.